(119d) The Deg-On System: Generation of a Cell-Based High Throughput Assay for the Screening of Proteasome Activators

The proteasome ubiquitin system (UPS) is a complex, multi-molecular machinery essential for the disposal of damaged proteins in eukaryotic cells. Discovered at the beginning of the 1980s by Aaron Ciechanover, Avram Hershko and Irwin Rose, who were rewarded with the 2005 Nobel Prize in Chemistry, it has been intensely studied both in mammalian and yeast cells. Particularly, impairment of UPS function has been associated with the development of a number of devastating human disorders, including Parkinson's and Alzheimer's diseases, which are characterized by inability of the UPS to cope with the aberrant accumulation of misfolded proteins. Unlike UPS inhibition, which has been long investigated and extensively characterized thanks to the availability of an array of small molecules that inhibit the proteasome through different molecular mechanisms, we currently have no means of enhancing proteasome activity, and more importantly, the molecular mechanisms that restrict proteasomal degradation remain unknown. The key requisite of a useful assay for the detection of enhancement of proteasomal activity is that the increase in degradation of a target protein must be linearly correlated with increase in a detectable and readily quantifiable signal. At present, no such strategy exists, as previous assay schemes relied on decrease in a signal tied to cellular protein accumulation. Such approaches are subject to a high degree of artifactual "false-negative" results. We recently developed a cell-based assay to allow correlation of UPS activity with a fluorescent output by coupling proteasomal degradation of a model substrate to the expression of the green fluorescent protein (GFP). This assay is particularly innovative as i) it allows us to correlate enhancement of proteasomal degradation with increase in an easily detectable and quantifiable signal, a strategy compatible with the development of a reliable high-throughput assay for the screening of chemical and genetic libraries, and ii) suboptimal concentrations of inducer molecule can be used to reach minimal activation of GFP expression, and restrict the assay signal window to the dynamic signal window to allow the detection of proteasome activators that induce minimal increase in degradation, possibly without perturbing other physiologic functions.